We investigated the ability of overexpression of the c-myc proto-oncogene to potentiate in vitro transformation by model chemical carcinogens. A mouse c-myc gene was introduced to C3H 10T1/2 and Rat 6 embryo fibroblast cell lines via a retroviral vector containing the gene for neomycin resistance. Our present work extends previous findings by showing that individual vectored C3H 10T1/2 clones have enhanced (twofold to sevenfold) sensitivity to benzo[a]pyrene (BP) and N-methyl-N-nitro-N′-nitrosoguanidine (MNNG). Rat 6 clones acquiring the c-myc gene display various degrees of altered morphology. They form orderly but densely packed cells, grow to higher saturation density, and yield microcolonies in soft agar. The degree of altered growth properties is directly correlated with the level of c-myc expression. Transient exposure of c-myc-expressing clones to BP and MNNG induced the formation of distinct, large colonies in soft agar, whereas the untreated cells formed microcolonies and the parental Rat 6 cells remained single cells in soft agar. We also demonstrated that the degree of responsiveness to chemical carcinogens of the clones correlates with their ability to form microcolonies in soft agar. These cells overexpressing c-myc may be used as a model system to study the interaction between oncogenes and chemical carcinogens in the process of multistage carcinogenesis. © 1992 Wiley-List, Inc.